Generation of islet β cell-specific Cre recombinase targeting vector by homologous recombination in bacteria
10.3724/SP.J.1008.2014.00185
- Author:
Ling LI
1
Author Information
1. Department of Pathophysiology, College of Basic Medical Sciences, Second Military Medical University
- Publication Type:Journal Article
- Keywords:
Cre recombinase;
Gene targeting;
Homologous recombination;
Insulin-secreting cells;
Internal ribosome entry site;
Targeting vector
- From:
Academic Journal of Second Military Medical University
2014;35(2):185-190
- CountryChina
- Language:Chinese
-
Abstract:
Objective To construct a knock-in targeting vector for expressing of Cre recombinase specifically in islet β cell and provide the key material for knock-in mice with Crr recombinase expressed in islet β cells, providing a knock-out animal model for studying the function of islet β cells. Methods In our study, we constructed a knock-in targeting vector using the third exon of insulin 2 (Ins2) as a target site with λ phage Red recombination system. Through a first homologous recombination, we cloned an about 12 kb genomic DNA fragment from the bacterial artificial chromosome (BAC) which contained Ins2 genomic DNA into a low copy vector pBR322-2s through gap repair. Meantime, a mini-targeting vector containing internal ribosome entry site (.IRES), DNA sequences encoding Crr recombinase and positive-negative-selection (PNS) gene was generated. After second recombination, the final Ins2-Cre targeting vector was generated. Results With the third exon of Ins2 used as the target, we successfully constructed the knock-in targeting vector expressing Cre recombinase which was controlled by endogenous Ins2 gene. Conclusion We have successfully constructed the knock-in targeting vector expressing Cre recombinase, it will provide an important material forcreating animal model of Cre recombinase which is specially expressed in islet β cells.
